Pentapartite fractionation of particles in oral fluids by differential centrifugation

Sachiko Matsumura,Masataka Kasahara,Yasutomo Yajima,Kiyotaka Shiba,Satoshi Yamamoto,Sadamitsu Hashimoto,Takeshi Nomura,Akira Katakura,Tamiko Minamisawa,Chiho Hiraga

doi:10.1038/s41598-021-82451-6

Sachiko Matsumura, Masataka Kasahara + Show 8 more

Open Access

https://doi.org/10.1038/s41598-021-82451-6

Copy DOI

Abstract

Oral fluids (OFs) contain small extracellular vesicles (sEVs or exosomes) that carry disease-associated diagnostic molecules. However, cells generate extracellular vesicles (EVs) other than sEVs, so the EV population is quite heterogeneous. Furthermore, molecules not packaged in EVs can also serve as diagnostic markers. For these reasons, developing a complete picture of particulate matter in the oral cavity is important before focusing on specific subtypes of EVs. Here, we used differential centrifugation to fractionate human OFs from healthy volunteers and patients with oral squamous cell carcinoma into 5 fractions, and we characterized the particles, nucleic acids, and proteins in each fraction. Canonical exosome markers, including CD63, CD9, CD133, and HSP70, were found in all fractions, whereas CD81 and AQP5 were enriched in the 160K fraction, with non-negligible amounts in the 2K fraction. The 2K fraction also contained its characteristic markers that included short derivatives of EGFR and E-cadherin, as well as an autophagosome marker, LC3, and large multi-layered vesicles were observed by electronic microscopy. Most of the DNA and RNA was recovered from the 0.3K and 2K fractions, with some in the 160K fraction. These results can provide guideline information for development of purpose-designed OF-based diagnostic systems.

Highlights

Oral fluids (OFs) contain small extracellular vesicles that carry disease-associated diagnostic molecules
Our focus has been on oral squamous cell carcinoma (OSCC), the predominant form of oral cancer, for the following reasons: (i) the number of patients with OSCC has been increasing[5] and (ii) the current staging of OSCC is incomplete and it frequently reoccurs in patients diagnosed at an early stage of O SCC6
We examined the distribution of extracellular nucleic acids by quantifying chromosomal DNA (SERPINA1; chromosome 14 and SLCO2B1; chromosome 11) and mitochondrial DNA (ND1 and N D340) using real-time PCR, indicating that both chromosomal and mitochondrial DNAs were abundant in the 0.3K and 2K fractions

Summary

Introduction

Oral fluids (OFs) contain small extracellular vesicles (sEVs or exosomes) that carry disease-associated diagnostic molecules. Molecules not packaged in EVs can serve as diagnostic markers For these reasons, developing a complete picture of particulate matter in the oral cavity is important before focusing on specific subtypes of EVs. Here, we used differential centrifugation to fractionate human OFs from healthy volunteers and patients with oral squamous cell carcinoma into 5 fractions, and we characterized the particles, nucleic acids, and proteins in each fraction. Previous work on EVs in this century have mostly focused on the smaller EV; recent studies have revealed that the larger EV have important biological activities[12,13,14,15,16,17,18] For this reason, knowledge of the complete range of EVs contained in OFs becomes important for developing OF-based diagnostic systems. The 2K and 10K fractions contained high-density (high contrast in TEM) particles, which likely represent oral bacteria cells (black arrows).

Objectives

Methods

Results

Conclusion